Class II (Ia) gene products play critical roles in a variety of T lymphocyte responses. They are the primary stimulating antigens in allogeneic and syngeneic mixed lymphocyte responses, they "restrict" recognition of foreign antigens by Lyt1+ T lymphocytes, and they control the ability of animals to respond to T-dependent antigens (immune response (Ir) gene function). A combination of immunological and molecular genetic approaches is being used to gain an understanding of the structural basis for this recognition of Ia molecules by T lymphocytes. Towards this goal we have cloned a cDNA corresponding to the AAlpha/Beta gene and utilized this probe to isolate complete Lambda genomic clones of AAlpha/Beta and Ak/Beta. These genes are being sequenced and used in DNA-mediated gene transfer experiments. Similar genomic clones corresponding to AAlpha; EAlpha, and EBeta are being isolated to provide reagents required for producing transfectants expressing intact two-chain Ia molecules. Mini-genomic libraries prepared from EMS derived immunoselected mutants of the TA3 (Ia+) B cell tumor line are being constructed to permit sequence comparison of the presumed variant Ak/Beta or Ak/Alpha genes to the wild type sequence, allowing identification of immunologically relevant regions of the Ia molecule. Regulatory sequences (promoters, enhancers) controlling the level of Ia expression will be identified by sequence and/or deletion mapping, and appropriate hybrid constructs involving inducible promoters will be made to permit us to selectively regulate the level of Ia in transfected cells. The data generated by these studies should be of significant help in enhancing our understanding of the critical structural features of Ia molecules recognized by T lymphocytes, possible sites on Ia molecules controlling interaction with antigen, and the importance of Ia density in T cell receptor occupancy and activation.